Discrete limbal epithelial stem cell populations mediate corneal homeostasis and wound healing
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PROVIDER: PRJNA705717 | ENA |
REPOSITORIES: ENA
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Project description:Stem cells (SCs) are traditionally viewed as rare, slow-cycling cells that follow deterministic rules dictating their self-renewal or differentiation. It was several decades ago, when limbal epithelial SCs (LSCs) that regenerate the corneal epithelium were among the first sporadic, quiescent SCs ever discovered. However, LSC dynamics, heterogeneity and genetic signature are largely unknown. Moreover, recent accumulating evidence strongly suggested that epithelial SCs are actually abundant, frequently dividing cells that display stochastic behavior. In this work, we combined single-cell RNA sequencing and advanced quantitative lineage tracing for in-depth analysis of the murine limbal epithelium. The generated data provides an atlas of cell states of the entire corneal epithelial lineage and reveales the co-existence of two novel LSC populations that reside in separate and well-defined sub-compartments. In the “outer” limbus, we discovered a primitive widespread population of quiescent LSCs (qLSCs) that uniformly express Krt15/Gpha2/Ifitm3/Cd63 proteins that serve as SC reservoire and in boundary formation. In the “inner” peri-corneal limbus, we identified prevalent active LSCs (aLSCs) that express Krt15-GFP/Atf3/Mt1-2/Socs3 and maintain homeostasis. We propose that these SC populations are abundant, follow stochastic rules and neutral drift dynamics. Notably, we provide evidence that T cells serve as niche cells for qLSCs, regulating quiescence and wound response. Taken together, we propose that divergent regenerative strategies are tailored to properly support tissue specific physiological constraints.
2021-03-02 | GSE167992 | GEO
Project description: Not available
| S-EPMC8254798 | biostudies-literature
Project description:Cornea, the outermost transparent layer of the eye, is the first line of defense against external threats. Following injury, the wound healing response is crucial to corneal repair and regeneration, yet its underlying mechanism is poorly understood. Our study was designed to investigate the role of dsRNA and its regulatory network in corneal wound healing.
2022-02-05 | GSE191228 | GEO
Project description:Transparent avascular cornea providing two third refraction to the eye. Restoration of corneal transparency and clear vision in a traumatic eye involves the action of many cytokines and signaling pathways. Out of several factors, stromal keratocytes/fibroblasts (CSFs) play a central role in corneal repair and wound healing. Post trauma, keratocytes/fibroblasts produce myofibroblasts to facilitate wound repair by synthesizing and secreting large extracellular matrix components, collagens, and alpha-smooth muscle actin stress fibers. This study aimed to perform RNASeq data analysis and pathway enrichments to gain a better understanding of gene regulation in corneal fibroblasts and myofibroblasts in corneal wound repair.
2024-03-11 | GSE260476 | GEO
Project description:Limbal vs. corneal epithelial basal cell gene expression patterns were identified and compared. Experiment Overall Design: 8 limbal and 8 corneal epithelial basal cells samples from 8 mice were dissected and mRNAs were isolated and amplified for microarray analysis
2007-11-22 | E-GEOD-4098 | biostudies-arrayexpress
Project description:Limbal vs. corneal epithelial basal cell gene expression patterns were identified and compared Keywords: repeat
2006-01-27 | GSE4098 | GEO
Project description:Limbal stem cells (LSCs), known as corneal epithelial stem cells, are located at the basal epithelial layer of the corneal limbus and serve an important function in maintaining the homeostasis of the corneal epithelium. Several putative molecular markers of LSCs have been previously identified. However, the specificity of these markers remains largely controversial. To address this gap in the current understanding of LSCs, we performed a transcriptome profiling of heterogeneous corneal limbal basal cells using single-cell transcriptomics technology to identify LSCs and their exclusive markers. We isolated limbal basal cells from two young donors, constructed scRNA-seq libraries, generated RNA-sequencing data using the 10x Genomics platform, and then finally obtained the transcriptome of about 18,000 individual single-cells using Cell ranger (https://10xgenomics.com). Next, we performed quality control, filtering and data integration using Seurat package (https://satijalab.org/seurat), about 16,400 cells were retained for further downstream analysis such as dimensional reduction, unsupervised clustering, and Differentially expressed gene selection, trajectory analysis and visualization. We identified 11 unique clusters of cells assigned to a putative cell type based on published known biomarkers for differentiation, proliferation and putative epithelial stem cells. As a results, we found 2 Terminally Differentiated Cell (TDC), 3 Post-Mitotic Cell (PMC), 1 Transient Amplifying Cell (TAC), 2 Limbal Progenitor Cell (LPC), Putative Limbal Stem Cell (LSC) and 2 Melanocyte (MC) sub-cell type clusters. Furthermore, we confirmed the trajectory order of LSC differentiation for 9 clusters using Pseudotemporal and Functional PCA analysis. Lastly, we validated each assigned cell subtypes and determined their location in human corneal limbus tissue with 9 markers using RNAscope We were able to reveal the heterogeneity of corneal limbal basal epithelium by defining novel dynamic trajectories for cell types in a pseudotemporal manner. This approach allowed us to identify the distinct clusters of LSCs and progenitors with exclusively expressed markers, and might apply for translational research on regenerating a normal corneal epithelium and restoring vision.
2021-01-14 | GSE153515 | GEO
Project description:Purpose: Single-cell RNA-sequencing (scRNA-seq) was used to interrogate the relatively rare stem (SC) and early transit amplifying (TA) cell populations in limbal/corneal epithelia from wild-type and autophagy-compromised mice. Results: Unbiased clustering detected 10 distinct populations: three clusters of mesenchymal and seven clusters of epithelial cells, based on their unique molecular signatures. A discrete group of mesenchymal cells expressed genes associated with corneal stromal SCs. We identified three limbal/corneal epithelial cell subpopulations designated as stem/early TA, mature TA, and differentiated corneal epithelial cells. Thioredoxin-interacting protein and PDZ-binding kinase (PBK) were identified as novel regulators of stem/early TA cell quiescence. PBK arrested corneal epithelial cells in G2/M phase of the cell cycle. Beclin1+/− mice displayed a decrease in proliferation-associated (Ki67, Lrig1) and stress-response (H2ax) genes. The most increased gene in beclin1+/− mice was transcription factor ATF3, which negatively regulates limbal epithelial cell proliferation. Conclusions: Establishment of a comprehensive atlas of genes expressed by stromal and epithelial cells from limbus and cornea forms the foundation for unraveling regulatory networks among these distinct tissues. Similarly, scRNA-seq profiling of the anterior segmental epithelia from wild-type and autophagy-deficient mice provides new insights into how autophagy influences proliferation in these tissues.
2020-09-24 | GSE158454 | GEO
Project description:Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing. Primary cultures of human corneal epithelial cells cultivated on BSA (number of replicates: 7), Collagen type I (number of replicates: 2), Collagen type IV (number of replicates: 2), Fibronectin (number of replicates: 2), Tenascin C (number of replicates: 2) and Laminin (number of replicates: 2) matrix. Central, internal and external ring of wounded Tissue-engineered human cornea.
2015-11-25 | E-GEOD-75336 | biostudies-arrayexpress
Project description:Limbal stem cells including epithelial and stromal/Mesenchymal stem cells that contribute to sustained corneal homeostasis, maintain their ability to act as self-renewal progenitor cells by virtue of their limbal niche and intercellular communication. Extracellular vehicles (EVs), including exosomes (Exos), are important paracrine mediators through their cargo transfer for intercellular communication in various stem cell niches. Previously we have shown the differential cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos, in limbal epithelial cells (LEC) in normal (N) and diabetic (DM) limbal niche. In the present study, to have a comprehensive knowledge of reciprocal LEC-LSC crosstalk, we investigated the proteomics and miRNA profile of exosomes derived from LEC and their regulatory roles in LSC in N and DM limbus. Our study showed wound healing and proliferation rates in primary N-LSC were significantly enhanced upon treatment by normal LEC-derived Exos (N-Exos), but not by diabetic Exos (DM-Exos). Further, N-Exos treated LSC showed downregulation of keratocyte markers, ALDH3A1 and lumican, but not keratocan, and upregulation of MSC markers, CD105, CD90, and CD73 compared to the DM-Exos treated LSC. Using next generation sequencing (NGS) and proteomics analysis, we revealed some miRNAs and proteins in the Exos that affect the cellular crosstalk and the function of the cornea. We also documented differences in DM vs. normal LEC-derived Exo’s cargos. Overall, DM-Exos have less effect on LSC proliferation, wound healing, and stem cell maintenance than N-Exos, likely by transferring their cargo proteins and/or regulatory miRNAs targeting cell cycle, ERK/MAPK, TGF-β, EMT, PI3K-Akt-mTOR signaling molecules. This suggests that the small RNA and protein cargo differences in DM vs. N LEC-derived Exos could contribute to the disease state. Our study revealed a complex contribution of Exos to health and diabetic state of corneal homeostasis and suggests the potential of EV therapeutics for diabetic cornea regenerative medicine
2023-10-23 | PXD040918 | Pride